IGEM:IMPERIAL/Protocols/T9002 AHL assay

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AHL assay from T9002 characterization

UNFINISHED !!!!

This is to clarify the protocol
-The Bacteria are measuring the concentration of AHL in the medium
-eg.
   If (as in the characterisation assay)you add 20ul of 1uM AHL to 1.98 ml of LB then
   you get a 10nM AHL solution The Bacteria will produce a GFP in response
   to this AHL concentration
-It is likely that the oscillations will be within the same order of magnitude as 
 the AHL assay. So to avoid the dilution problem of adding supernatant from the sample
 we wish to test, to an assay culture, thereby diluting the AHL to an undetectable
 concentration it makes much more sense to re-suspend the T9002 cells in the test supernatant,
 thereby solving the dilution problem. This is what this protocol tries to achive
 JohnChattaway 08:15, 22 August 2006 (EDT)


Motivation

This protocols is designed to measure an unknown concentration of AHL in a medium.

Materials & Equipment

  • Equipment
    • Wallac Victor 3 Multi-Well Fluorimeter
    • Ependorf Tubes
    • Gilson Pippettes
    • 37oC Shaker
  • Materials
    • Solution to assay
    • GFP Standard Solution
    • E.coli Growth Medium w/Ampicilin (LB/M9)
    • E.coli Culture Containing T9002

Protocol

Prepare T9002 cells:

  • Inoculate a culture from 10ul of stored T9002 in 2ml LB/M9 growth medium containing 50ug/ml Ampicilin.
  • Incubate at 37oC for overnight in a shaker.
  • Following day, prewarm LB/M9 to 37oC by placing in the 37oC incubator
  • Measure and record OD600_1 in report sheet
  • Inoculate a 8ml fresh culture from the o/n to bring back the OD600 to 0.1, use prewarmed LB/M9 + Ampicilin.
    • Volume used to inoculate new culture = (0.1/OD600_1)*8ml
  • Return LB/M9 to incubator
  • Incubate new culture at 37oC for 2 hours in a shaker - This returns cells to exponential phase
  • Measure and record the OD600_2 in report sheet
  • Dilute again for an OD of 0.1 in a new culture of 15ml of a prewarmed LB/M9 + Ampicilin.
    • Volume used to inoculate new culture = (0.1/OD600_2)*15ml
  • Vortex new T9002 culture.

Prepare medium to assay:

  • Spin down solution to assay and retreive supernatant
  • Spin down T9002 solution and get rid of the supernatant - This avoids a dilution effect when measuring AHL
  • Resuspend T9002 cells into solution to be measured.

AHL incubation and GFP reading

  • Pipette samples into a 96 well plate
  • Add 200uL of growth medium to a well to act as a control
  • Incubate 96 well plate in a 37oC shaker for 4 hours - Allows GFP expression can reach steady state
  • After the 4 hours add 200uL of 200x diluted GFP standard solution
  • Take a reading
  • Taking readings
    • Take the plate from the water bath
    • Use the Victor3 to measure flourescence and absorbance
    • Return the plate to the water bath
  • Take 3 further readings every 20 minutes - This is to assess whether GFP has in fact reached steady state


Any questions, see Tom.


Many thanks to Drew Endy and Barry Canton from MIT for providing the protocol on which this is based.

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